An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion)

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ZooKeys 416: 113-155 (2014)

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An unexpected clade of South American ground
beetles (Coleoptera, Carabidae, Bembidion)

David R. Maddison!

| Department of Integrative Biology, Oregon State University, Corvallis, OR 97331, USA

Corresponding author: David R. Maddison (david.maddison@science.oregonstate.edu)

Academic editor: 77 Assmann | Received 12 April 2014 | Accepted 23 May 2014 | Published 17 June 2014
http://zoobank.org/CE0561FB-5 EE4-498 B-A2C2-EDF9B14F241D

Citation: Maddison DR (2014) An unexpected clade of South American ground beetles (Coleoptera, Carabidae,
Bembidion). ZooKeys 416: 113-155. doi: 10.3897/zookeys.416.7706

Abstract

Phylogenetic relationships of the Antiperyphanes Complex of the genus Bembidion are inferred using DNA
sequences from seven genes (two nuclear ribosomal, four nuclear protein coding, and one mitochondrial
protein coding). Redefined subgenera within the complex are each well-supported as monophyletic. Most
striking was the discovery that a small set of morphologically and ecologically heterogeneous species
formed a clade, here called subgenus Nothonepha. This unexpected result was corroborated by the discov-
ery of deep pits in the lateral body wall (in the mesepisternum) of all Nothonepha, a trait unique within
Bembidion. These pits are filled with a waxy substance in ethanol-preserved specimens. In one newly dis-
covered species (Bembidion tetrapholeon sp. n., described here), these pits are so deep that their projections
into the body cavity from the two sides touch each other internally. These structures in Bembidion (No-
thonepha) are compared to very similar mesepisternal pits which have convergently evolved in two other
groups of carabid beetles. The function of these thoracic pits is unknown. Most members of subgenus
Nothonepha have in addition similar but smaller pits in the abdomen. A revised classification is proposed

for the Antiperyphanes Complex.

Keywords
Carabidae, Bembidiini, Bembidion, phylogeny, systematics, DNA, South America, ground beetles

Copyright David R. Maddison. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

114 David R. Maddison / ZooKeys 416: 113-155 (2014)

Introduction

Ground beetles of the genus Bembidion are distributed throughout the temperate re-
gions of the world (Maddison 2012). The fauna of South America is diverse, with
about 140 species described (Jeannel 1962; Toledano 2002; Toledano 2008), mostly
occurring in cooler, southern regions of the continent, and northward in the Andes.
Although many species resemble northern-hemisphere subgenera scattered throughout
the two major clades of Bembidion (the Bembidion Series and the Ocydromus Series), all
known species in South America are in fact members of only three groups within the
Bembidion Series: the subgenera Notaphus and Nothocys, as well as the Antiperyphanes
Complex (Maddison 2012).

The Antiperyphanes Complex is a clade restricted to South and Central America.
Eight subgenera are considered to belong to the complex (Maddison 2012; Maddi-
son et al. 2013): Antiperyphanes Jeannel, Antiperyphus Jeannel, Chilioperyphus Jeannel,
Plocamoperyphus Jeannel, Nothonepha Jeannel, Pacmophena Jeannel, Notholopha Jean-
nel, and Ecuadion Moret and Toledano, with two subgenera suspected of belonging
(Notoperyphus Bonniard de Saludo (1969), Pseudotrepanes Jeannel (1962)). Members
of this complex are moderately diverse in form, and are typically shades of brown,
orange, and yellow, although a few species have metallic colors (Figs 1-3) They are
abundant along edges of bodies of water (rivers, creeks, ponds, lakes, snowfields, and
ocean; Figs 4A,B) in south temperate regions (especially Argentina, Chile, Peru, and
Bolivia), and at higher elevations from Patagonia north into Central America. In the
mountains of Ecuador, Peru, and nearby areas, one group (subgenus Ecuadion) has
radiated into alpine grasslands (Fig. 4C), cloud forest leaf litter (Fig. 4D), clay cliffs,
and other habitats distant from open water.

Although monophyly of the Antiperyphanes Complex is well supported (Maddison
2012), details about its phylogenetic structure are poorly known. Only 20 species of
the 95 or so known species have been included in phylogenetic studies, with some key
taxa missing. For example, only two species of what is considered the heterogeneous
subgenus Antiperyphus have been sampled, and its type species (B. philippii Germain)
has not previously been examined. Similarly, only two of the more than 50 species of
Ecuadion were included in previous studies.

The current more in-depth investigation into phylogeny of the Antiperyphanes
Complex was inspired by discovery, on the gravel shores of Rio Puntra on Isla
Grande de Chiloé, Chile, of a large, distinctive, undescribed species of Bembidion
(Figs 5A and 5B). This unusual species appeared to fall outside any named subge-
nus, and is given the name Bembidion tetrapholeon in this paper. In order to infer
its relationships, additional members of the Antiperyphanes Complex were gathered
and sequenced. Preliminary results from the sequences of one gene indicated the
existence of a clade so surprising that I initially considered it fallacious, a result of
errors in sample labeling, but when additional samples and genes provided stronger
support, that explanation was no longer tenable. This apparent clade, including the

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 115

Figure |. Adults of subgenera Antiperyphanes and Chilioperyphus. A Bembidion (Antiperyphanes) rufo-
plagiatum, Argentina: Neuquén: Arroyo Quefi at Lago Queni, DRM voucher V100796 B B. (Antipery-
phanes) hirtipes, Argentina: Mendoza: Pampa Palauco, DRM voucher V100792 C B. (Antiperyphanes)
spinolai, Argentina: Chubut: Rio Azul at Lago Puelo, DRM voucher V100788 D B. (Antiperyphanes)
zanettii, Ecuador: Napo: Rio Quijos W of Baeza, DRM voucher V100791 E B. (Antiperyphanes) man-
dibulare, Chile: Reg. X, Chiloé: Cucao, DRM voucher V100789 F B. (Chilioperyphus) orregoi, Argentina:
Chubut: Rio Azul at Lago Puelo, DRM voucher V100674. Scale bar 1 mm.

new species, consisted of taxa that are much more diverse in form (Fig. 5) and
habitat (Fig. 6) than other small clades of similar molecular diversity. This paper
reports the results of sequencing of seven genes which together provide very strong
support for this clade. The discovery of the clade led to the search for morphologi-
cal synapomorphies of its members, and a striking, derived character was found in
thoracic structure. Although the focus of the paper is on this unexpected clade, the
relationships of other members of the Antiperyphanes Complex are explored, and a
new classification is proposed for the group.

116

David R. Maddison / ZooKeys 416: 113-155 (2014)

C .

Figure 2. Adults of subgenera Antiperyphus and Notholopha. A Bembidion (Antiperyphus) philippii, Argentina:
Neuquén: Rio Collén Cura ca 13 km S La Rinconada, DRM voucher V100787 B B. (Notholopha) scitu-
lum, CHILE: Reg. VII: Los Niches E of Curicé, DRM voucher V100790 € B. (Notholopha) sexfoveolatum,
CHILE: Reg. IX: 16.3 km E Malalcahuello, Cuesta Las Raices, DRM voucher V100598. Scale bar 1 mm.

Methods

Specimens examined and depositories. Specimens examined are from or will be de-

posited in the collections listed below. Each collection’s listing begins with the coden
used in the text.

BMNH
CMNH
CTVR
EMEC
IADIZA
MACN

MNHN
MNNC
NHMW
OSAC
USNM
ZMUC

The Natural History Museum, London, UK

Carnegie Museum of Natural History, Pittsburgh, Pennsylvania, USA
Luca Toledano collection, Verona, Italy

Essig Museum Entomology Collection, University of California, Berkeley, USA
Instituto Argentino de Investigaciones de las Zonas Aridas, Mendoza, Argentina
Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos
Aires, Argentina.

Muséum National d’ Histoire Naturelle, Paris, France

Museo Nacional de Historia Natural, Santiago, Chile

Naturhistorisches Museum, Wien, Austria

Oregon State Arthropod Collection, Oregon State University, Corvallis, USA
National Museum of Natural History, Washington, USA

Natural History Museum of Denmark, University of Copenhagen, Copen-

hagen, Denmark

Collecting methods. Specimens were collected by hand or using an aspirator; speci-
mens were found during the day in their habitat after splashing the soil with water, or with

the aid of a headlamp at night, when the beetles are more actively moving on the surface.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 117

F/

Figure 3. Adults of subgenus Ecuadion. A Bembidion chimborazonum, ECUADOR: Pichincha: Paso de
la Virgen, DRM voucher V100793 B B. sanctaemarthae, ECUADOR: Napo: Rio Chalpi Grande, DRM
voucher V100798 C B. andersoni, ECUADOR: Pichincha: Reserva Yanacocha, start Andean Snipe Trail,
DRM voucher V100655 D B. walterrossi, ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, DRM vouch-
er V100794 E B. cotopaxi, ECUADOR: Pichincha: km 17 on route 28 W of Papallacta DRM voucher
V100797 F B. ricei, ECUADOR: Napo: Rio Chalpi Grande, DRM voucher V100622. Scale bar 1 mm.

Most specimens were killed and preserved in Acer sawdust to which ethyl ace-
tate was added. Specimens collected specifically for DNA sequencing were killed and
stored in 95% or 100% ethanol, with best results obtained when the abdomen was
slightly separated from the rest of the body to allow better penetration, or when the
reproductive system was dissected out through the rear of the abdomen within a few
minutes of the beetle’s death in ethanol. Ethanol was decanted and refilled at least two
times within the first few weeks after death. Storage was then at 4° or -20°C.

Taxon sampling for DNA studies. DNA was newly sequenced from 25 species of
the Antiperyphanes Complex of Bembidion; to these sequences were added sequences
of 20 more species of this complex acquired during previous studies (Table 1). Forty
additional species of Bembidion were also included in the analyses (27 species of the

David R. Maddison / ZooKeys 416: 113-155 (2014)

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120 David R. Maddison / ZooKeys 416: 113-155 (2014)

Figure 4. Habitats of the Antiperyphanes Complex. A River shore, Argentina: Neuquén: Rio Collén
Cura, about 13 km S La Rinconada, 625m. On the sandy bank in the foreground Bembidion philippii
is abundant, as is B. (Nothonepha) sp. nr. lonae. Also occurring on the sand banks are B. orregoi and B.
(Nothonepha) eburneonigrum. On the upper sand banks across the river are B. mandibulare, and in the
gravel are B. spinolai B Edges of snowfields at Chile: Reg. IX: Volcan Lonquimay, 1910m. Habitat of B.
(Notholopha) sexfoveolatum © Open high-elevation grassland at Ecuador: Pichincha: Paso de la Virgen,
4060m, habitat of three species of subgenus Ecuadion: Bembidion chimborazonum, B. guamani, and B.
humboldti D Leaf litter in cloud forest, Ecuador: Pichincha: Reserva Yanacocha, 0.1152°S, 78.5837°W,
3540m, habitat of Bembidion andersoni, B. georgeballi, and B. onorei.

Bembidion Series exclusive of the Antiperyphanes Complex, plus 13 species of Bem-
bidion outside the Bembidion Series). The 27 additional Bembidion Series species are
evident in Fig. 7; details about these species and specimens sequenced are available in
Maddison (2012). The 13 species outside of the Bembidion Series included are Bem-
bidion variegatum Say, B. planum (Haldeman), B. stephensi Crotch, B. tetracolum Say,
B. hastii Sahlberg, B. punctulatum Drapiez, B. properans (Stephens), B. concolor (Kir-
by), B. chalceum (Dejean), B. lunulatum (Geoffroy), B. wickhami Hayward, B. genei
illigeri Netolitzky, and B. cf. exquisitium Andrewes.

Nine additional specimens of Bembidion tetrapholeon sp. n., were sequenced (Table 2)
to examine variation. The 10 specimens sequenced in total included specimens from two
localities in Chile and three localities in Argentina (Tables 1, 2), and included the typical

uniformly black specimens, and others that have a large orange spot on their elytra.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 121

Table 2. Additional sampling of Bembidion tetrapholeon to examine DNA sequence variation. The DNA
voucher number is listed in the “#” column. Color of elytra: BL: nearly black; OR: black with large orange
spot. * indicates the holotype. Further details about the localities are provided under the description of B.
tetrapholeon and in the Appendix 1. All of these are “genseq-2” sequences (Chakrabarty et al. 2013), except

for DNA2356 (genseq-1) and DNA1752 (genseq-3).

# Color CAD we ArgK COI

1752 BL KJ653110 | KJ653213 KJ653143
2356* BL KJ653112 | KJ653215 | KJ653082 KJ653145
2357 OR KJ653113 | KJ653216 | KJ653083 KJ653146
2562 BL KJ653115 | KJ653217 | KJ653084 KJ653148
2566 BL KJ653119 | KJ653218 | KJ653085 KJ653152
2555 OR KJ653114 KJ653147
2564 OR KJ653117 KJ653150
2565 BL KJ653118 KJ653151
2563 BL KJ653116 KJ653185 | KJ653053 | KJ653149

Vouchers are housed in the David Maddison voucher collection at Oregon State
University, with the exception of voucher number DNA2356, the holotype of B.
tetrapholeon, which is deposited in LADIZA.

DNA sequencing. The genes studied, and abbreviations used in this paper, are:
28S or 28S rDNA: 28S ribosomal DNA; 18S or 18S rDNA: 18S ribosomal DNA;
ArgK: arginine kinase; CAD: carbamoyl! phosphate synthetase domain of the rudimen-
tary gene; COI: cytochrome oxidase I; Topo: topoisomerase I; wg: wingless.

Fragments for these genes were amplified using the Polymerase Chain Reaction on
an Eppendorf Mastercycler Thermal Cycler ProS, using TaKaRa Ex Taq and the basic
protocols recommended by the manufacturer. Primers and details of the cycling reac-
tions used are given in Maddison (2012). The amplified products were then cleaned,
quantified, and sequenced at the University of Arizona’s Genomic and Technology
Core Facility using a 3730 XL Applied Biosystems automatic sequencer.

Assembly of multiple chromatograms for each gene fragment and initial base calls
were made with Phred (Green and Ewing 2002) and Phrap (Green 1999) as orches-
trated by Mesquite’s Chromaseq package (Maddison and Maddison 2011a; Maddi-
son and Maddison 2011b) with subsequent modifications by Chromaseq and manual
inspection. Multiple peaks at a single position in multiple reads were coded using
IUPAC ambiguity codes.

Sequences of COI for two species showed evidence of nuclear copies of this mito-
chondrial gene (“numts”) (Thalmann et al. 2004). For B. georgeballi (voucher 2661)
and B. walterrossii (voucher 2650), sequences obtained using the LC1490-HC2198
primer pair yielded different sequences from the B1490-Bcoi2R primer pair. The
former had numerous double-peaks, suggesting that the sequences included numts
(Maddison 2012); the LC1490-HC2198 sequence for B. walterrossii also had one stop
codon near its 5’ end. The reads from the B1490-Bcoi2R primers are much cleaner
and show no double-peaks. Although all of these sequences have been submitted to

122 David R. Maddison / ZooKeys 416: 113-155 (2014)

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Figure 5. Adults of subgenus Nothonepha. A Bembidion tetrapholeon (black form), Argentina: Neu-
quén: Arroyo Quenhi at Lago Quefi, DRM voucher V100781 B B. tetrapholeon (orange form), Argentina:
Chubut: Rio Azul at Lago Puelo, DRM voucher V100780 C B. germainianum, Argentina: Neuquén:
Rio Salado at route 40, DRM voucher V100782 D B. /onae, Argentina: Mendoza: Salinas del Diamante,
DRM voucher V100786 E B. eburneonigrum, Argentina: Neuquén: Rio Neuquén at Chos Malal, DRM
voucher V100785 F B. tucumanum, Argentina: Mendoza: Salinas del Diamante, DRM voucher V100783
G B. engelhardti engelhardti, Argentina: Neuquén: Rio Salado at route 40, DRM voucher V100784. Scale

bar 1 mm.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 123

GenBank, only the sequences from the B1490-Bcoi2R primers have been included in
the analyses.

Alignment and data exclusion. The appropriate alignment was obvious for all
protein-coding genes. There were no insertion or deletions (indels) evident in the sam-
pled CAD, ArgK, Topo, or COI sequences. In wingless, there were two small, well-
separated indels, restricted to only three taxa: three inserted nucleotides in Bembidion
(Zemetallina) parviceps Bates, and six inserted nucleotides in a different region in the
two species of subgenus Omotaphus Netolitzky sampled. These inserted nucleotides
were excluded from analyses.

The ribosomal genes showed a slightly more complex history of insertions and
deletions. Both genes were first subjected to multiple sequence alignment in MAFFT
version 7.130b (Katoh and Standley 2013), using the L-INS-i search option and oth-
erwise default parameter values. Visual inspection suggested no needed improvements,
and no ambiguously aligned regions that required exclusion.

Molecular phylogenetic analysis. Models of nucleotide evolution were chosen
with the aid of jModelTest version 2.1.1 (Darriba et al. 2012; Guindon and Gascuel
2003) (for each gene) and PartitionFinder version 1.1.1 (Lanfear et al. 2012) (for parts
of the partition chosen by PartitionFinder). Among the models supported by RAxML,
the model chosen for all genes by the Bayesian Information Criterion was GTR+L+I.

Likelihood analyses of nucleotide data were conducted using RAxML version 7.2.6
(Stamatakis 2006). Analyses were conducted on each gene individually, as well as a
matrix of seven genes concatenated together. Two different partitioning schemes were
examined: (1) with seven parts, one for each gene; (2) as chosen using the Bayesian
Information Criterion (BIC) using PartitionFinder (Lanfear et al. 2012). The partition
chosen by BIC contained three parts: one part with third positions of COI; a second part
with third positions of the nuclear protein-coding genes; a third part with all remaining
sites. For bootstrap analyses 2000 replicates were conducted; maximum likelihood boot-
strap (MLB) values are reported as percentages. In addition to these bootstrap analyses,
searches for maximum likelihood trees were conducted using 1000 search replicates.

Most-parsimonious trees (MPT) were sought using PAUP* (Swofford 2002). To
search for most parsimonious trees, 2000 replicates were conducted, each beginning
with a starting tree formed with the random addition sequence option, with each
replicate saving no more than 25 trees. For parsimony bootstrap analyses in PAUP*,
1000 bootstrap replicates were examined, each of which used a heuristic search with
four replicates, each beginning with a starting tree formed by the random addition se-
quence option, with TBR branch rearrangement, with each replicate saving no more
than 25 trees; the estimated bootstrap values are reported as parsimony bootstrap
percentages (PB).

Morphological methods. General methods of specimen preparation for morpho-
logical work, and terms used, are given in Maddison (1993; 2008). After dissection
from the body, genitalia were prepared by treatment in 10% KOH at 65 °C for 10
minutes followed by multi-hour baths of distilled water, 5% glacial acetic acid, dis-
tilled water, and then ethanol. Male genitalia, when studied, have been mounted in

124 David R. Maddison / ZooKeys 416: 113-155 (2014)

[Se Fg aPC ee S *

Figure 6. Habitats of the subgenus Nothonepha. A Habitat of B. (Nothonepha) eburneonigrum (on sand
patches) and B. (Nothonepha) sp. nr. lonae (on sand patches and in gravel). This habitat (Chile: Reg. IX:
Rio Allipén at route 119, 132m) is also home to Bembidion spinolai Solier and B. rufoplagiatum B Habi-
tat of B. (Nothonepha) tucumanum, B. (Nothonepha) lonae, and B. (Nothonepha) engelhardti (Argentina:
Mendoza: Salinas del Diamante, 1280m). The beetles are common under rocks and around vegetation
on the salt-encrusted clay and sand banks of this saline pond; in the same habitat B. (Notaphus) cillenoides
Jensen-Haarup and two other Notaphus are common C On the sand shores of this desert river B. (otho-
nepha) germainianum, B. (Nothonepha) engelhardti, and B. (Nothonepha) lonae are common (Argentina:
Neuquén: Rio Salado at route 40, 725m) D Type locality of B. (Nothonepha) tetrapholeon (Argentina:
Neuquén: Arroyo Queni at Lago Quefi, 830m). The beetles are found under rocks along the river shore;
B. rufoplagiatum is also common in this habitat.

Euparal between two small coverslips attached to archival-quality heavyweight water-
color paper. Measurements for body length are from apex of the labrum to apex of the
longer elytron.

An examination of external and internal features of the exoskeleton was conduct-
ed to search for possible apomorphies of one of the discovered clades, focusing on
externally-visible pits in the mesothorax and anterior region of the abdomen. Internal
skeletal elements were studied on specimens whose soft tissue was dissolved by placing
the opened body in a 10% KOH solution at 65 °C for 10 minutes.

Studies of muscles and other internal soft tissue were conducted on specimens killed
and preserved in 100% ethanol. This is not the ideal preservation medium, as it leaves

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 125

muscles brittle and more difficult to trace. However, since the unexpected discovery of
structures requiring internal examination, better-fixed specimens have not been available.

Photographs of body parts were taken with a Leica Z6 and JVC KY-F75U camera.
For pronotal, elytral, and genitalic images, a stack of photographs at different focal
planes was taken using Microvision’s Cartograph software; these TIFF images were
then merged using the PMax procedure in Zerene Systems’s Zerene Stacker; the final
images thus potentially have some artifacts caused by the merging algorithm.

Data resources

Sequences have been deposited in GenBank with accession numbers KJ653019 through
KJ653220. GenBank numbers for the two apparent numts sequences are KJ653155
for DNA2661 and KJ653156 for DNA2650. Aligned data for each specimen as well
as files containing inferred trees for each gene and concatenated matrices are available
in Suppl. material 1 and 2, and have been deposited in the Dryad Digital Repository,
http://doi.org/10.5061/dryad.47r16.

Results

Molecular results

The inferred phylogeny is presented in Figs 7 and 8, with support values for notable
clades given in Table 3. Maximum likelihood trees and maximum likelihood bootstrap
trees for each gene and the concatenated matrices are illustrated in Suppl. material 3
and 4. They are also contained in the NEXUS file S1 in the Suppl. material 1.

The monophyly of each subgenus (indicated by color in Figs 7 and 8) is well sup-
ported by analyses of the concatenated matrix (MLB=100 and PB=100 for all but
one subgenus) and at least four genes (Table 3), except for subgenus Antiperyphanes.
Antiperyphanes is monophyletic in the maximum likelihood trees of four genes, but
bootstrap support is low (Table 3).

The basal split in the Antiperyphanes Complex appears to be between clade b and
clade d (Fig. 8). Clade b is strongly supported in seven-gene analyses (MLB=100,
PB=100), and there is bootstrap support in individual analyses of four genes (Table 3).
As a subgenus of Bembidion, this clade would take the name Nothonepha Jeannel, as it
contains Bembidion lonae, the type species of Nothonepha. Clade d is also strongly sup-
ported in the seven-gene analyses (MLB=100, PB=99), and there is moderate to weak
bootstrap support from four genes (Table 3).

Within the Antiperyphanes Complex, strongly supported relationships between
subgenera include a clade containing Antiperyphanes and Chilioperyphus, and a sister-
group relationship between that clade and the subgenus Ecuadion (Fig. 8, Table 3). B.
(Antiperyphus) philippii appears as the sister group to subgenus Notholopha.

David R. Maddison / ZooKeys 416: 113-155 (2014)

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An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 127

Bembidion solieri
Bembidion cupreostriatum
Bembidion sp. nr. ugartei
Bembidion calverti
Bembidion posticale
Bembidion cillenoides
Bembidion rapidum
Bembidion aratum
Bembidion scintillans
Bembidion flohri
Bembidion dorsale
Bembidion varium
Bembidion salinarium
Bembidion elizabethae
Bembidion frontale
Bembidion fortestriatum
Bembidion pseudocautum
Bembidion assimile
Bembidion muscicola
Bembidion (Zecillenus) sp. 1
Bembidion ateradustum
Bembidion errans
Bembidion proprium
Bembidion maorinum
Bembidion tairuense
Bembidion anchonoderum
Bembidion hokitikense
Bembidion parviceps
Bembidion (Omotaphus) sp. 1
Bembidion (Omotaphus) sp. 2
Bembidion poculare
Bembidion sciakyi
Bembidion quadrimaculatum
Bembidion quadripustulatum
Bembidion jacksoniense
Bembidion niloticum
Bembidion foveolatum
Bembidion riverinae
Bembidion articulatum
Bembidion ephippium
Bembidion octomaculatum
Bembidion transversum
Bembidion diligens
Bembidion vile
Bembidion constricticolle
Bembidion cf. cognatum
Bembidion affine
Bembidion versicolor
Bembidion (Nothocys) sp. 1
Bembidion anthracinum
Bembidion (Nothocys) sp. 2
Bembidion marginatum
Bembidion tetrapholeon
hag ater Ap Ure Oe a
embidion lonae
Bembidion sp. nr. lonae Nothonepha
Bembidion germainianum
Bembidion engelhardti
Bembidion tucumanum A
Bembidion philippii Antiperyphus
Bembidion sexfoveatum
Bembidion (Notholopha) sp. "Lago Roca"
Bembidion rugosellum Notholopha
Bembidion stricticolle
Bembidion scitulum
Bembidion (Notholopha) sp. “Nahuelbuta"
Bembidion melanopodum
Bembidion orregoi ii
Bembidion sp. "Cal" Chilioperyphus
Bembidion mendocinum
Bembidion caoduroi
Bembidion sp. nr. caoduroi
Bembidion zanettii
Bembidion mandibulare 2
Bembidion neodelamarei Antiperyphanes
Bembidion spinolai
Bembidion chilense
Bembidion sp. nr. chilense
Bembidion hirtipes
Bembidion rufoplagiatum
Bembidion agonoides
Bembidion chimborazonum
Bembidion paulinae paulinae
Bembidion rogersi
Bembidion sanctaemarthae
Bembidion (Ecuadion) sp. "Mendoza”
Bembidion guamani m
Bembidion (Ecuadion) sp. "Papallacta” Ecuadion
Bembidion jimburae
Bembidion walterrossii
Bembidion georgeballi
Bembidion andersoni
Bembidion rawlinsi
Bembidion onorei
Bembidion ricei
—_ Bembidion cotopaxi
Bembidion humboldti

Antiperyphanes Complex

Figure 7. Tree of highest likelihood found for the combined, seven-gene matrix with partitioning scheme
chosen by PartitionFinder. Only members of the Bembidion Series shown; more distant outgroups not

depicted. Scale bar: 0.01, branch lengths as reconstructed by RAxML.

128 David R. Maddison / ZooKeys 416: 113-155 (2014)

(b) Bembidion tetrapholeon
Bembidion eburneonigrum
Bembidion lonae
is © de 100 Bembidion sp. nr. lonae Nothonepha
95 /48 Bembidion germainianum
98/92 Bembidion engelhardti
100 Bembidion tucumanum :
© Bembidion philippii Antiperyphus
Bembidion scitulum
Bembidion sexfoveatum Notholopha
100 /98 (f) Bembidion stricticolle
Bembidion (Notholopha) sp. “Lago Roca"
100 Bembidion rugosellum
Bembidion (Notholopha) sp. "Nahuelbuta”
96/92 Bembidion melanopodum
Bembidion sp. "Cal" a4
@ Bembidion mendocinum Chilioperyphus

100 Bembidion orregoi
100/99 (h) D

98/94
100

Bembidion caoduroi
Bembidion sp. nr. caoduroi
100 Bembidion zanettii
Bembidion mandibulare
Bembidion neodelamarei Antiperyphanes
&) pores 100 Bembidion spinolai ae
100 Bembidion chilense
100 Bembidion sp. nr. chilense
98/97 Bembidion hirtipes
® 100 98/98 Bembidion rufoplagiatum
(m) Bembidion agonoides
Bembidion chimborazonum
100 98/96 Bembidion paulinae paulinae
97/91 Bembidion rogersi Ecuadion
99/96 Bembidion sanctaemarthae
100 Bembidion (Ecuadion) sp. "Mendoza"
Bembidion guamani
(n) 100 Bembidion (Ecuadion) sp. "Papallacta"
Bembidion andersoni
100 Bembidion georgeballi
Bembidion rawlinsi
Bembidion jimburae
100 Bembidion walterrossii
Bembidion onorei
Bembidion ricei
99/100 Bembidion cotopaxi
100/96 Bembidion humboldti

Figure 8. Maximum likelihood bootstrap tree showing only those clades appearing in 90% of the boot-
strap replicates; taxa outside of the Antiperyphanes Complex not shown. Numbers below branches indicate
maximum likelihood bootstrap percentage / parsimony bootstrap percentage. Circled letters on branches

correspond to groups documented in Table 3.

As a whole, the Antiperyphanes Complex is supported as monophyletic (clade a in
Figs 7 and 8, Table 3), although not as strongly as in an earlier study with more limited
sampling of the group (Maddison 2012). Individual gene support for the clade is only
provided by CAD and to a lesser extent wingless (Table 3), but the concatenated analy-
ses have MLB=88 and PB=86. Lack of monophyly of the complex in some analyses
(e.g., individual gene analyses of 28S and 18S) is a result of Nothonepha falling in the
Bembidion Series separate from the rest of the complex.

Different analytical methods yielded similar results for the concatenated, seven-
gene matrices. [he two partition schemes examined (by gene and as chosen by Parti-
tionFinder) resulted in maximum likelihood trees that differ only in the placement of
Bembidion georgeballi within subgenus Ecuadion. In maximum likelihood bootstrap
analyses, clades with MLB>90 were the same in both partition schemes. Parsimony
analyses showed similar results to maximum likelihood (Table 3).

Within Bembidion tetrapholeon sp. n., the 10 specimens sequenced from five lo-
calities showed little variation in DNA, and the variation observed was not correlated

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 129

with presence of an orange spot. There was no variation observed in CAD (n=10),
ArgK (n=5), Topo (n=10), and 28S (n=10), over a total of more than 3090 bases. In
the wingless gene (n=6) variation was observed at three third-position sites, all of which
represent synonymous differences, and for each of which some other specimens were
heterozygous for the variants. COI showed the most variation with variability at eight
sites, seven of which represented synonymous differences and one a non-synonymous
difference. At seven of these sites, nine of the ten specimens had the same nucleotide,
with the tenth specimen being unique; the specimen that was unique varied from site
to site. The most distinct specimen was DNA2236, from Chiloé, with three unique
nucleotides in the more than 650 bases of COI sequenced.

Morphological results

With the unexpected discovery that B. tetrapholeon sp. n., belongs in a clade with an
assortment of morphologically and ecologically diverse Bembidion, the search for syna-
pomorphies for this clade became compelling.

Mesothoracic pits. The most striking derived feature observed was presence in all
Nothonepha species of a pit in each lateral wall of the mesothorax. This mesepisternal
pit (Fig. 9A) appears empty in many specimens killed in ethyl acetate, but in most
specimens preserved in ethanol, a waxy substance is visible within it (Fig. 9A). When
extracted and placed in glycerin on a microscope slide, this substance appears slightly
yellowish-gray and contains no obvious substructure or particles (including no evident
bacteria or fungal spores) when examined at 400x with transmitted, brightfield light
(n=2, from B. tetrapholeon). In contrast, all other members of Bembidion examined to
date lack such a pit (e.g., Fig. 9B).

In B. tetrapholeon these paired structures, one on either side, internally manifest as
large intrusions which touch in the center of the body cavity (Figs 10A, C, E). Typi-
cal Bembidion have no such structures internally (Figs 10B, D, F). There is variation
within Nothonepha in the size of the intrusions, with B. engelhardti having relatively
small intrusions (and thus relatively shallow pits) (Fig. 11).

Examination of musculature in B. tetrapholeon (n=4) and B. tucumanum (n=1)
revealed no muscles attached to the internal intrusions, although the course of some
muscles appeared to be bent by the necessity to wrap around the structures. There
were no evident large glands associated with the intrusions, although there were small
patches of tissue on their internal surfaces.

Two other groups of carabids reported to have mesepisternal pits were also ex-
amined, members of subgenus Tachylopha of the genus Elaphropus (Bruneau de Miré
1966; Erwin 1970) and the genus Oodinus (Spence 1982). I have specimens of these
preserved in 95% ethanol, and both have large pits in the mesepisternum in the same
place as Bembidion (Nothonepha). In Elaphropus (Tachylopha) leleupi Basilewsky from
South Africa the pits are filled with a waxy substance similar to that seen in Nothonepha
(Fig. 12A). Internally these pits appear as two large intrusions that join in mid-thorax

130 David R. Maddison / ZooKeys 416: 113-155 (2014)

elytron

3

Figure 9. Left lateral region of the prothorax and mesothorax; top of each photograph is anterior. msst:
mesosternum; msepst: mesepisternum; msep: mesepimeron (note that the boundaries between these scle-
rites are not evident externally in A. A Bembidion (Nothonepha) tetrapholeon, DRM voucher V100810
B B. (Antiperyphanes) zanettii, DRM voucher V100811. Scale bar 0.1 mm.

to form a tunnel (Fig. 12B); in the two specimens I have dissected there is no evidence
of a septum at the point of joining, and the waxy substance fills the tunnel. I have seen
ethanol-preserved specimens of E. (Tachylopha) basilewskyi Bruneau de Miré from Ga-
bon (identified with Bruneau de Miré (1966)) and E. (Tachylopha) spenceri (Sloane)
from Australia (identified with Baehr (1988)) that have similar pits also filled with a
waxy substance. The apparently related subgenus Sphaerotachys also has mesepisternal
pits, although they are much smaller than those seen in Tachylopha; a specimen |

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 131

cs intrusions

Figure 10. Mesothorax, dorsal surface and soft tissue removed. msst: mesosternum; msepst: mesepister-
num; msep: mesepimeron. Scale bar 0.1 mm. (A, C and E) Bembidion (Nothonepha) tetrapholeon, DRM
voucher V100766 B, D, and F B. (Antiperyphanes) zanettii, DRM voucher V100767 A, B oblique ven-

tral view; view from lower left side, slightly in front of mesothorax. C, D oblique dorsal view; view from

upper right side, slightly behind the mesothorax. E, F anterior view.

132 David R. Maddison / ZooKeys 416: 113-155 (2014)

Figure | 1. Mesothorax, dorsal surface and soft tissue removed, of Bembidion (Nothonepha) engelhardti
rayoda Yoledano. Oblique dorsal view; view from upper right side, slightly behind the mesothorax. Scale

bar 0.1 mm.

prothorax

mesothorax NY

elytron

Figure 12. Mesothoracic structures of Elaphropus (Tachylopha) leleupi Basilewsky. A Left lateral region
of the prothorax and mesothorax B Anterior view of mesothorax, dorsal surface and soft tissue removed.
Scale bars 0.1 mm.

have examined from Hans Merensky Nature Reserve, Republic of South Africa, has
pits similar to those shown in Fig. 11. The single ethanol-preserved Oodinus alutaceus
(Bates) (identified with Bousquet (1996)) that I have examined, from south Texas, also
has mesepisternal pits, but internally the intrusions do not touch, and are more similar

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 133

oe 8B. (Nothonepha) tetrapholeon ; Ps B. (Antiperyphanes) mandibulare me

$ -_ . elytron

Metacoxa

Figure 13. Ventral surface of anterior end of abdomen and posterior region of metathorax. The sec-
ond and third abdominal segments are marked by II and IH. A Bembidion tetrapholeon, DRM voucher
V100773. B B. mandibulare, DRM voucher V100772 C B. sp. nr. lonae, DRM voucher V100771 D B.
lonae, DRM voucher V100770. Scale bar 0.1 mm.

in structure to those of B. (Nothonepha) than E. (Tachylopha). In some specimens of
Oodinus, the pits are also filled with a waxy substance (Spence 1982).

Abdominal pits. In additional to mesepisternal pits, B. tetrapholeon has a promi-
nent pit on each side of the abdomen, ventrally, between abdominal segments I and
III (Fig. 13A). In ethanol-preserved specimens, this pit is filled with a waxy substance
similar to that in the mesepisternal pits. Almost all other species of subgenus Notho-
nepha have similar pits (e.g., B. (Nothonepha) sp. nr. lonae, Fig. 13C); they are lacking
only in B. (Nothonepha) lonae (Fig. 13D), the sister to B. sp. nr. lonae.

Internally these abdominal pits are evident as knob-shaped intrusions (Fig. 14A, C).
Consistent with the lack of externally visible pits, B. /onae lacks these intrusions, and has in-
stead only a ridge between the abdominal segments (Fig. 14D), as is typical in Bembidion.

Outside of Nothonepha | have seen no species of Bembidion with as prominent ab-
dominal pits, and most species lack them entirely. B. (Antiperyphanes) mandibulare, for
example, lacks abdominal pits and has only a slight linear depression in that region (Fig.
13B), and internally a simple ridge is evident (Fig. 14B). There is much variation between
members of the Antiperyphanes Complex in this feature, however, with some species hav-
ing an evident pit/internal intrusion (e.g., B. (Antiperyphus) philippii, Fig. 14E, and B.
(Antiperyphanes) zanettii), and others (e.g., B. (Ecuadion) chimborazonum, Fig. 14F) hav-
ing a low, wide hump internally. Outside of the Antiperyphanes Complex all species exam-
ined either have a sinuate (Fig. 14G) or straight (Fig. 14H) ridge internally in this region.

Whatever internal structure is present between abdominal segments I] and HI,
whether a ridge, or low mound, or knob-like intrusion, in the species examined this

134 David R. Maddison / ZooKeys 416: 113-155 (2014)

B. (Nothonepha) tetrapholeon

B. (Antiperyphanes) mandibulare 3

B. (Nothonepha) sp.nr. lonae
sp

7. Ve

%

intrusion

B. (Antiperyphus) philippii B. (Ecuadion) chimborazonum

B. (Zeplataphus) charile

SP membrane

ridge

Figure 14. Inner surface of the anterior portion of the abdomen, soft tissue removed. The abdomen is
slightly tilted to the left. sp: sclerotized patch on membrane that bounds the front of the abdomen on the
ventral side. All species shown are members of the Bembidion Series; A-F are members of the Antipery-
phanes Complex; A, C and D are members of the subgenus Nothonepha A Bembidion tetrapholeon, DRM
voucher V100766 B B. mandibulare, DRM voucher V100805 C B. sp.nr. lonae, DRM voucher V100803
D B. lonae, DRM voucher V100802 E B. philippiiz, DRM voucher V100804 F B. chimborazonum, DRM
voucher V100809 G B. charile Bates, DRM voucher DNA1171 H B. quadrimaculatum oppositum Say,
DRM voucher V100807. Scale bar 0.1 mm.

structure serves (at least in part) as an apodeme. In B. tetrapholeon (n=5), for example,
a muscle bundle is attached to the apex of the internal intrusion, and extends forward
to the small sclerotized patch (sp in Fig. 14C) in the membrane that serves as the front

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 135

boundary above the ventral margin of the abdomen. Another muscle bundle extends
from this sclerotized patch forward and laterally to the lateral wall of the body, where it
attaches to an external rod-like sclerite that is connected to the posterior lateral corner
of the metanotum; this rod-like sclerite extends posterior laterally from that point to
near the metepimeron. I have examined B. (Ecuadion) chimborazonum (n=1) and it has
a similar muscle attached to the low mound in the same region; B. (Bracteon) foveum
Motschulsky (n=1), B. (Ocydromus Complex) nebraskense (n=2), and the bembidiine
Lionepha casta (n=2) all have a similar muscle connecting the intersegmental ridge at
the equivalent region to the small sclerotized patch.

Discussion

Monophyly of Nothonepha. DNA data strongly support Nothonepha as a clade.
Four genes (CAD, weg, 28S, 18S) independently have bootstrap support for the clade
(Table 3), and the concatenated seven-gene analysis has MLB=100 and PB=100.
Combined with the striking synapomorphy of mesothoracic pits, evidence for this
clade becomes convincing. As Darwin (1859) noted, “We may err in this respect in
regard to single points of structure, but when several characters, let them be ever so
trifling, occur together throughout a large group of beings having different habits,
we may feel almost sure, on the theory of descent, that these characters have been
inherited from a common ancestor.”

The unexpectedness of Nothonepha. The relationship between species here
grouped into subgenus Nothonepha was so unexpected when first discovered from se-
quences of 28S that it was dismissed, and considered to be the result of DNA contami-
nation or mislabeled extractions. This small clade, of only ten known species, includes
some of the largest Bembidion in South America (B. germainianum, up to 6.4 mm in
length, Fig. 5C), and some of the smallest (B. /onae, down to 2.4 mm in length, Fig.
5D). They range in habitat from cobbles shores of small, cold, clear rivers (Fig. 6D)
to mixed shores of large rivers (Fig. GA), and sand shores of desert rivers (Fig. GC) to
warm, exposed salt flats (Fig. 6B). In the field, beetles in this group give the appearance
of rather different groups of carabids. In my first field encounter with live B. lonae, I
mistook them for tachyines of the genus Elaphropus Motschulsky; B. germainianum is
reminiscent of the Nearctic Bembidion perspicuum Casey, a member of the Ocydromus
Series of Bembidion; B. eburneonigrum looks very much like a small member of the
subgenus Notaphus as it scurries on the sandy shores of rivers.

There are many examples of clades throughout the tree of life that contain species
of diverse forms living in diverse habitats. Why then is Nothonepha unexpected? The
current classification, to the extent that it might be a predictor of relationships, would
suggest that these taxa are not related. B. /onae is the only described species of those
sampled that was placed in Nothonepha; the very similar but undescribed B. sp.nr. lonae
would have been placed there as well. The other described species (B. germainianum,
B. engelhardti, B. tucumanum, B. eburneonigrum, and B. tucumanum) had all been

136 David R. Maddison / ZooKeys 416: 113-155 (2014)

classified in subgenus Antiperyphus, along with B. philippii. When I first discovered
B. tetrapholeon, | thought it represented a separate lineage requiring a new subgeneric
name, as it is very different in form from any other South American Bembidion. This
apparently added a third element to the diverse group. However, the current classifica-
tion, constructed with limited data and without phylogenetic analyses, may not be the
best predictor of phylogenetic relationships.

Nonetheless, Nothonepha is a morphologically heterogeneous group (Fig. 6). I have
been studying Bembidion systematics for over three decades, and whatever predictive
map my brain has developed from all the data accumulated over the years contained
no hint that the species shown in Fig. 6 formed a clade.

However, it may not be the diversity of form within Nothonepha that is unusual,
but rather the diversity given the lack of intermediate forms and small size of the group.
Other clades in the South American fauna are also diverse in form and size (e.g., Ec-
uadion, Fig. 3), but they have many more species, some of which are intermediate be-
tween the more distinct forms. The lack of intermediate forms in Nothonepha may be
a result of low speciation rates with high rates of morphological evolution, or it might
be a result of extinction of intermediate forms; it is unlikely to be a lack of sampling,
as enough collecting has been done in South America to suggest that there are not a
large number of undescribed species of Nothonepha. A full investigation of patterns of
morphological and molecular rates, times of divergence, and speciation and extinction
rates relative to other clades is beyond the scope of this paper, but would be a worth-
while topic for future studies.

Function of mesepisternal pits. Exoskeletal invaginations are widespread in bee-
tles (Grebennikov and Leschen 2010). These cavities occur on many different body
parts, including in the lateral regions of the mesothorax (e.g., in the ptiliid tribe Dis-
cheramocephalini (Grebennikov 2008; 2009)); their function probably varies from
group to group. Many have been thought to be mycangia for storing fungal spores,
but this is well documented in only two of the many independent origins of such cavi-
ties (Grebennikov and Leschen 2010). In a few groups of polyphagan beetles (e.g.,
the Staphylinoidea subfamily Scydmaeninae, the Cucujoid families Cyclaxyridae and
Nitidulidae, the Tenebrionoidea family Zopheridae) there are species with pits con-
taining a waxy substance (Grebennikov and Leschen 2010). Wax has been proposed
to act as a defensive shield (Lawrence and Hlavac 1979), or as a medium for retaining
fungal spores (Grebennikov and Leschen 2010).

The functions of mesepisternal pits in the two carabid groups in which they were
previously described is not known, but functions have been hypothesized. Erwin (1970)
proposes that pits of Elaphropus (Tachylopha) are insertion points for ant mandibles, al-
lowing ants to carry the adults around. Bousquet (1996) calls the structures in Oodinus
“apodemal pits”, which implies a function as internal attachment points for muscles.

The similarities between the wax-filled mesepisternal pits observed in the bembi-
diine Bembidion (Nothonepha), the tachyines Elaphropus (Tachylopha) and Elaphropus
(Sphaerotachys), and the oodine Oodinus are striking (e.g., Figs 9A and 12A in this
paper, and Fig. 145 in Bousquet (1996)), enough so that a common function might

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 137

be hypothesized. Although the shape and nature of the deepest pits and largest intru-
sions differs between the groups (e.g., there are no Nothonepha known with the merged
intrusions of some Tachylopha), the less extreme forms are indistinguishable externally
and internally. ‘These features are surely convergent, as the clades are not closely related.
Bembidion and Elaphropus are both members of the subfamily Trechinae, but they are
each deeply nested within independent clades (Maddison and Ober 2011); Oodinus is
a member of the tribe Oodini (Bousquet 1996; Spence 1982), which is nested within
several clades in the superfamily Harpalinae, itself a well-supported clade (Maddison
et al. 1999; Ober and Heider 2010). It may be that these pits serve different functions
in these clades, but consistency of structure suggests they may be serving the beetles in
similar ways in the three different groups.

Correlates in way of life might provide some hints about function. Members of the
three carabid groups are all presumably generalist predators, as is typical in Carabidae
(Thiele 1977). They are also all terrestrial, but associated with shorelines. Bembidion
(Nothonepha) species occur at edges of bodies of water in southern South America.
Elaphropus (Tachylopha) occurs in similar habitats. I have seen Elaphropus (Tachy-
lopha) spenceri (Sloane) (identified using Baehr (1988)) from multiple localities in
Queensland, Australia, labeled as being found at “water’s edge” along creek shores.
Bruneau de Miré (1966) reports numerous species along rivers, and states that they
can be abundant in swamps and moist forest humus. Oodinus occurs at the edges of
marshes and swamps (Bousquet 1996; Spence 1982). However, many other carabid
groups lacking these pits are also found in these habitats, including many other Bem-
bidion, Elaphropus, and Oodini.

It appears unlikely that mesepisternal pits are used as ant handles in Bembidion
(Nothonepha) and Oodinus, and probably not in Elaphropus (Tachylopha). Erwin’s
(1970) hypothesis was based in part on the unusual elytral structure of Tachylopha,
which is narrow above the mesepisternum and which possesses a notch into which
the base of ant mandibles could fit. Although most Nothonepha have narrow-enough
pronota to allow curved, sickle-shaped mandibles of a large ant access to the pits, that
seems much less likely for Oodinus, which are wide-bodied, oval carabids. Oodinus have
a distinct ledge along the lateral edge of their bodies; to fit a mandible tip into the pit
underneath this ledge an ant would need exceptionally curved mandibles. In general,
ground-nesting ants are relatively rare in wet, near-shore, seasonally inundated habi-
tats, and one would not expect large ants (ground-nesting or arboreal) with sufficiently
curved mandibles in these near-shore environments (Philip S. Ward, pers. comm.). I
have observed ants only rarely in the near-water habitats of subgenus Nothonepha, and
not at all in the case of the four localities at which I have collected B. tetrapholeon or
the three localities at which I have found B. germainianum. Furthermore, there is no
evidence for any association between ants and these three carabid groups.

There is also evidence against the mesepisternal pits functioning as apodemes. As
noted above, Nothonepha and Tachylopha do not have muscles attached to the internal
walls of the mesepisternal pits. Spence (1982) reports that there are no conspicuous
muscles that attach to the internal walls of the pits in Oodinus.

138 David R. Maddison / ZooKeys 416: 113-155 (2014)

It is possible that the function is as a reservoir for the wax, although where the wax
is produced is not evident. Spence (1982) states that the pit walls are perforated by nu-
merous channels in Oodinus; these might be ducts for glandular secretions. I have not
detected any large glands internally near the intrusions, although there is a thin layer of
tissue in places on the inner surface of the structures. It is also possible that the wax is
not produced near the intrusion; it might be produced elsewhere on the beetle’s body,
and in fluid form flow into the pits. (As noted above, the wax is not evident in many
ethyl acetate killed specimens, but rather in ethanol killed specimens, suggesting that
the substance may precipitate in ethanol, but exist as a liquid otherwise.) More detailed
histological work is needed to explore possible glandular sources.

Whatever the source of the wax, its function (if it has one) is unclear. It seems
unlikely that it would be for retaining fungal spores (Grebennikov and Leschen 2010),
as there is nothing known about these generalist predators that would suggest a benefit
to the beetle to retain fungal spores. Wax as a defensive coating is plausible (Lawrence
and Hlavac 1979), but more studies are needed to explore this and other possibilities.

Function of abdominal pits. In contrast to the mesepisternal pits, the abdominal
pits in Bembidion (Nothonepha) evidently serve (in part) as apodemes, that is, as attach-
ment points for muscles. However, the function of those muscles is unclear; in general
the nature and function of muscles at the junction of the metathorax and abdomen in
beetles is poorly known (Rolf Beutel, pers. comm. 2014). Even if the abdominal pits
serve as apodemes, they may serve additional functions as well, perhaps related to the
presence of wax that appears similar to that found in the mesepisternal pits.

Taxonomic treatment

Three clades of Bembidion comprise the South American fauna: subgenus Notaphus
Dejean (32 species (Jeannel 1962; Toledano 2002; Toledano 2008)), subgenus Notho-
cys Jeannel (13 species (Toledano 2002; Toledano 2008)), and the Antiperyphanes
Complex. Along with a number of lineages outside of South America, these all belong
to one subclade of Bembidion, the Bembidion Series (Maddison 2012). Subgenus No-
taphus is widespread and abundant throughout the New World, with over 50 species
known from North America; in addition, seven species occur in the Old World (Lobl
and Smetana 2003). Nothocys is restricted to southern South America, occurring in
Chile, Argentina, Peru, and Bolivia (Toledano 2002; Toledano 2008). The largest of
these clades is the Antiperyphanes Complex, with about 95 described species (Jeannel
1962; Maddison 2012; Toledano 2002; Toledano 2008), and many undescribed.

Antiperyphanes Complex

Remarks. Members of the Antiperyphanes Complex are diverse in form (Figs 1-3, 5).

There are no recognized derived morphological characteristics of the group, although

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 139

the clade is moderately well supported by the concatenated DNA sequence data (Fig. 8,
Table 3). Within the South American fauna, most species can be recognized by the
lack of an N sclerite in the internal sac of the male genitalia (Toledano 2008). How-
ever, some members of the complex, including some species in subgenus Nothonepha,
have a small sclerite that could be homologous to the N sclerite (Toledano 2008).

The Antiperyphanes Complex, as here classified, consists of at least five subgenera:
Antiperyphanes, Chilioperyphus, Antiperyphus, Notholopha, Ecuadion, and Nothonepha.
Each of these subgenera is briefly discussed below, with notes about their composi-
tion. Two other poorly known subgenera, Pseudotrepanes Jeannel and Notoperyphus
Bonniard de Saludo, are likely members of this complex, but specimens will need to be
examined to confirm their membership.

Subgenus Antiperyphanes Jeannel, 1962

Antiperyphanes Jeannel, 1962; type species Bembidium spinolai Solier, by original des-
ignation.

Plocamoperyphus Jeannel, 1962; type species Bembidium mandibulare Solier, by original
designation. New synonymy.

Remarks. ‘This group contains at least 19 described species (Toledano 2002; Toledano
2008), and is characterized by males having an aedeagus lacking a brush sclerite, and
with a very long flagellum (Maddison et al. 2013; Toledano 2008).

Included here are some species previously placed in subgenus Antiperyphus by
Jeannel (1962): B. hirtipes Jeannel), B. ringueleti (Jeannel), B. rufoplagiatum Ger-
main, B. uniforme Csiki, and B. parvum (Jeannel). B. mandibulare Solier belongs to
Antiperyphanes as well, and is nested well within it (Fig. 8); thus, subgenus Plocamop-
eryphus is a synonym of Antiperyphanes. There are no morphological characteristics
of B. mandibulare that would suggest it is not a member of Antiperyphanes; it shares
all apomorphies of the group. As first reviser, I choose Antiperyphanes as the valid
name of the group.

Antiperyphanes has two distinct clades, each very well supported: the B. caoduroi
group (clade j in Fig. 8; Fig. 1D), consisting (among the sampled species) of three large
species from the northern Andes (Fig. 1D); (2) the remaining Antiperypanes (clade k in
Fig. 8; Figs LA—C, E). Each is supported by MLB=100 and PB=100 in the multi-gene
analyses, and individually by support from five to seven genes (Table 3). As a whole,
however, the monophyly of Antiperyphanes is only weakly supported by the combined
analysis and analyses of four genes (Table 3).

Members of this subgenus are found at the edges of bodies of water. For exam-
ple, B. rufoplagiatum and B. zanettii are common on gravel and cobble river shores,
B. ringueleti is found on gravel and sand shores of smaller creeks, and B. mandibu-
lare on the sand beaches of the Pacific Ocean in Chile and sand beaches of rivers
in Argentina.

140 David R. Maddison / ZooKeys 416: 113-155 (2014)

Subgenus Chilioperyphus Jeannel, 1962

Chilioperyphus Jeannel, 1962; type species Bembidium orregoi Germain, by original
designation.

Remarks. ‘This subgenus contains two described species (Jeannel 1962; Maddison et
al. 2013) (Fig. 1F) and at least three undescribed species. Males are characterized by
having a brush sclerite, and by having an extremely elongate flagellum, so long that it
can only fit within the median lobe through folding (Maddison et al. 2013). Members
of this subgenus occur on steep sand or clay banks of rivers and creeks.

Subgenus Antiperyphus Jeannel, 1962

Antiperyphus Jeannel, 1962; type species Bembidium philippii Germain, by original

designation.

Remarks. As noted by Toledano (2008), Jeannel’s concept of Antiperyphus was poly-
phyletic, with at least B. hirtipes, B. ringueleti, B. rufoplagiatum, and B. uniforme Csiki
belonging within Antiperyphanes. This is confirmed in part by my results (Fig. 8, Ta-
ble 3). In addition, B. engelhardti, B. eburneonigrum, B. tucumanum, and B. germaini-
anum are members of Nothonepha, not Antiperyphus. Of the species included in the
subgenus by Jeannel (1962), this leaves only the type species, B. philippii (Fig. 2A).

In addition, Bembidion peterseni Jensen-Haarup (1910), from Mendoza, Argentina,
can tentatively be placed here. I have examined a male syntype (in ZMUC), and it is
similar in appearance to B. philippii, although with much deeper and longer elytral striae.
It is not a member of Nothonepha (as it lacks mesepisternal pits), nor is it a member of
Antiperyphanes (it has a brush sclerite, and does not have the long flagellum characteristic
of Antiperyphanes). The internal sac of the male genitalia, although difficult to see because
of the nature of the preparation, appears very similar to that of B. philippii.

B. philippii is common on sand shores of rivers in the provinces of Neuquén and
Chubut in Argentina; it also occurs in Chile.

Subgenus Notholopha Jeannel, 1962

Notholopha Jeannel, 1962; type species Bembidium punctigerum Solier, by original
designation.

Pacmophena Jeannel, 1962; type species Bembidium scitulum Erichson, by original
designation.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 141

Remarks. Notholopha consists of 11 described species (Toledano 2002; Toledano
2008), and several undescribed (two of which are sequenced here). These are small
beetles with large, protruding eyes (Fig. 2B,C), with small flagella in the internal sac
of the male genitalia, and with brush sclerites. They have the general appearance when
running of a member of the Holarctic subgenus Bembidion. Some frequent habitats
similar to those of subgenus Bembidion, including dry habitats far from water (B.
stricticolle), or upper banks of creeks (e.g., B. sp. “Nahuelbuta”). Others occur at high
elevation near small rivulets in open, alpine areas (e.g., B. rugosellum and B. melanopo-
dum), or at the edges of snowfields (B. sexfoveolatum).

Jeannel considered Pacmophena and Notholopha to be two subgenera within the
genus NVotholopha. As the characters that Jeannel used to distinguish the two are minor
characters such as surface texture and antennal length, and as it appears that Pacmo-
phena is paraphyletic with respect to the Notholopha (s. str.) (Fig. 7), I consider them
synonymous, with Notholopha as the valid name.

Subgenus Ecuadion Moret & Toledano, 2002

Ecuadion Moret & Toledano, 2002; type species Bembidion fulvocinctum Bates, by
original designation.

Remarks. Ecuadion is the largest subgenus in the Antiperyphanes Complex, with over
50 described species (Maddison and Toledano 2012; Moret and Toledano 2002; To-
ledano 2008; Vigna Taglianti and Toledano 2008) and likely many undescribed. It
occurs from Costa Rica south to the mountains near Mendoza, Argentina. There are
no known exoskeletal synapomorphies of the group, but it is well-supported by the
molecular data, with bootstrap support in six of the seven genes examined (Table 3).

Ecuadion falls into two distinct clades among the sampled species: (1) the B. chim-
borazonum group (clade m in Fig. 8), consisting mostly of larger, long-legged species
(Figs 3A, B); (2) remaining Ecuadion (clade n in Fig. 8), consisting of mostly smaller
species with shorter appendages (Figs 3C-—F). The B. chimborazonum group is sup-
ported by all genes examined (Table 3); support for the complementary clade is not
quite as strong, with the clearest evidence coming from ribosomal genes (Table 3).

Unlike most Bembidion, this subgenus has radiated in habitats away from water.
Some occur in leaf litter in cloud forests (e.g., B. andersoni, B. georgeballi, B. onorei, B.
sp. “Papallacta”; Fig. 4D), in habitats that would typically be occupied by the genus
Trechus Clairville in North America. A number of species are found in open, high-el-
evation grasslands (e.g., B. chimborazonum, B. guamani, B. humboldti; Fig. 4C). Some
species occur on the upper banks of creek shores (e.g., B. sanctaemarthae, B. ricei); oth-
ers are found on clay or silt cliffs (e.g., B. agonoides, B. walterrossii).

142 David R. Maddison / ZooKeys 416: 113-155 (2014)

Subgenus Nothonepha Jeannel, 1962

Nothonepha Jeannel, 1962; type species Bembidium baptisatum Csiki (=B. lonae Jensen-
Haarup), by original designation.

Remarks. As here defined, the subgenus Nothonepha includes all species of the Antiperyphanes
Complex possessing mesepisternal pits. Seven described species belong to Nothonepha:
Bembidion lonae Jensen-Haarup, 1910 (Fig. 5D)
B. pallideguttula Jensen-Haarup, 1910
B. eburneonigrum Germain, 1906 (Fig. 5E)
B. engelhardti Jensen-Haarup, 1910
B. engelhardti engelhardti Jensen-Haarup, 1910 (Fig. 5G)
B. engelhardti rayoda Toledano, 2008
B. tucumanum (Jeannel, 1962) (Fig. 5F)
B. germainianum Toledano, 2002 (Fig. 5C)
B. tetrapholeon Maddison, sp. n. (Figs 5A, B)

Four of these species (B. germainianum, B. tucumanum, B. engelhardti, and B. eburneo-
nigrum) were formerly placed in subgenus Antiperyphus. In addition, there are at least
three undescribed species (including B. sp. nr. /onae, sequenced here). ‘The species figured
by Toledano (2008) as B. germainianum is an undescribed species related to B. germaini-
anum. A revision of the subgenus is in preparation (Roig-Jufent and Maddison).

Bembidion (Nothonepha) tetrapholeon sp. n.
http://zoobank.org/90188564-6B1E-4F0E-B41 1-5AD99047F716
Figs 5A, B, 9A, 15, 16, 17A, 18

Holotype male (IADIZA), with 3 labels: “Argentina: Neuquén: Arroyo / Quefi at
Lago Quefi, 830m, / 40.1575°S 71.721°W, / 10-11.ii.2007. DRM 07.035. / D.R.
Maddison, S.A.Roig”, “David R. Maddison / DNA2356 / DNA Voucher” [print-
ed on pale green paper], and “HOLOTYPE / Bembidion / tetrapholeon / David R.
Maddison” [printed on red paper]. Genitalia in glycerine in small plastic vial beneath
specimen; extracted DNA stored separately. GenBank accession numbers for DNA
sequences of the holotype are KJ653049 (28S), KJ653145 (COD), KJ653112 (CAD),
KJ653181 (Topo), KJ653215 (wg), and KJ653082 (Argk).

Paratypes. Total of 244, in IADIZA, MACN, MNNC, OSAC, MNHN,
BMNH, EMEC, CTVR, and CMNH, from “Argentina: Neuquén: Arroyo / Que-
fi at Lago Queni, 830m, / 40.1575°S, 71.721°W, / 10-11.ii.2007. DRM 07.035.
/ D.R. Maddison, S.A.Roig” [135 exx.], Argentina: Neuquén: Rio Pichi / Traful nr
Lago Traful, 810m, / 40.4867°S, 71.5958°W, / 12.ii.2007. DRM 07.039. / D.R.
Maddison, S.A.Roig” [95 exx], “Argentina: Chubut: Rio / Azul at Lago Puelo, 200m
/ 42.0929°S, 71.6244°W / 13.11.2007. DRM 07.044. / D.R. Maddison” [12 exx],

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 143

Figure 15. Pronotum of Bembidion tetrapholeon, DRM voucher V100781. Scale bar 0.1 mm.

“Argentina: Chubut: Rio / Azul at Lago Puelo, 200m / 42.0929°S, 71.6244°W, /
13.ii.2007. DRM 07.045. / S.A.Roig, D.R. Maddison” [1 exx].

Additional material examined. CHILE: Reg. X, Chiloé: Rio Puntra at rt 5,
55m, 42.1661°S, 73.7256°W, 19.i.2006. DRM 06.075. D.R. Maddison [5 exx,
OSAC, MNNC]. CHILE: Region X, Rio Pullinque at Puente Huanehue, 8 km NE
Panguipulli. 16 Jan 2002, 39.6162°S, 72.2286°W, 1590 ft. W. D. Shepard [2 exx,
OSAC].

Additional identified material. The following specimens have been examined by
Luca Toledano and confirmed to belong to this species (based upon photographs we
have shared). CHILE: Reg. X, Los Lagos, P.N. Vicente Péres Rosales, Petrohué, Lago
Todos los Santos, 190m, mouth Rio El Caulle, 41.0924°S, 72.3950°W. 5.i.2014.
L. Toledano, R. Olivieri, J.P. Morales. [1 ex, CTVR]; CHILE: Region XI, Parque
Nat. Rio Simpson, H. Franz [4 exx, NHMW]; CHILE: Reg. X, I. Chiloé, R. Punta,
31.1.1986. M. Spies. [1 ex, USNM].

Type locality. Argentina: Neuquén: Arroyo Quefi at Lago Quefi, 830m,
40.1575°S, 71.7210°W. The habitat at the type locality is a cobble, gravel, and coarse
sand river shore (Fig. 6D); the river is cold and has crystal-clear water. In the same
habitat members of the genus Bembidarenas Erwin are abundant, as is Bembidion (An-
tiperyphanes) rufoplagiatum.

Derivation of specific epithet. From the Greek “tetra”, meaning “four”, and
“pholeon”, meaning “pit”, referring to the four prominent pits visible on the underside
of adults.

144 David R. Maddison / ZooKeys 416: 113-155 (2014)

Figure 16. Humeral region of left elytron and posterior corner of pronotum of three Bembidion (Notho-
nepha) species. A Bembidion tetrapholeon, DRM voucher V100781 B B. lonae, DRM voucher V100786
C B. germainianum, DRM voucher V100782. Arrows show the anterior end of the lateral elytral groove
and bead. Scale bar 0.1 mm.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 145

Diagnosis. A large, sleek, shiny Bembidion, with an unusual body form (Figs
5A,B) of narrow forebody and large elytra. With its shape, color, and smoothness it is
one of the most distinctive Bembidion species in South America, and no other known
species is likely to be confused with it; it is more reminiscent of some species in New
Zealand, e.g., B. (Zeplataphus) dehiscens Broun (Lindroth 1976).

Length (4.7—5.7 mm, with most specimens above 5.0 mm). Color piceous (Fig.
5A), with legs and antennae in some specimens slightly paler, and with a few speci-
mens having a large orange spot just in front of the elytral apices (Fig. 5B).

Head with shallow and parallel frontal furrows.

Pronotum narrow, cordate, with hind angles flaring outward (Fig. 15). Very
smooth, without punctures, and with a linear basolateral foveae; without distinct ca-
rina at hind angle. Lateral bead of pronotum not complete, not reaching front angle
of prothorax and only in some specimens reaching the hind angle. One midlateral and
one basolateral seta on each side.

Each elytron with two discal setae (ed3 and ed5); ed3 in third stria. Elytral striae
with prominent punctures in their basal half, but with striae 2—7 absent in about the
hind 40% or more of the elytra, such that the posterior discal seta, ed5, is in a region
without striae. Striae 7 absent in many specimens. In many specimens the striae are ef-
faced anteriorly, especially striae 2 and 3. Lateral bead of elytron effaced anteriorly, not
extended onto shoulder (Fig. 16A), similar to that of B. (Vothonepha) lonae (Fig. 16B),
but unlike most other Bembidion (e.g., B. (Nothonepha) germainianum, Fig. 16C).

Mesothorax with prominent pits in the mesepisternum (Fig. 9A), which appear
internally as large intrusions that touch in the middle (Figs 10A, C, E). Smaller pits are
present ventrally at the junction of abdominal segments II and III (Fig. 13A), which
are evident internally as knob-like intrusions (Fig. 14A).

Hind wings full.

Microsculpture absent from entire dorsal surface of the body except for the cervical
region of the head, labrum, and faintly on the clypeus; the beetles are thus brilliantly
shiny. Microsculpture absent from most of the ventral surface as well, with the most
notable microsculpture being on the undersurface of the head.

Aedeagus with nearly straight ventral margin, tip of variable width (Fig. 17).
Prominent brush sclerite, and with flagellum not clearly evident from the left side.
There is no evident correlation between aedeagal structure and presence or absence of
orange spots on the elytra (compare Figs 17A, B to Figs 17C, D).

Morphological variation. The most noted variation is in color of the elytra. Of
the 257 specimens examined (including the six specimens identified by Luca Toleda-
no), 245 have uniformly piceous elytra (Fig. 5A); the remaining 12 have a large, diffuse
orange spot occupying most of the posterior third of the elytra (Fig. 5B). Ten of these
orange-spotted specimens are from the three localities in Argentina, with at least one
orange-spotted specimen from each locality, and two of the orange-spotted specimens
are from Chile. In most of orange-spotted specimens, the posterior discal seta (ed5) is
in the orange region, but immediately around the seta is a small dark spot. No other

146 David R. Maddison / ZooKeys 416: 113-155 (2014)

Figure 17. Male aedeagus of Bembidion tetrapholeon. A Black form, Chile: Region X, Rio Pullinque at
Puente Huanehue, 8 km NE Panguipulli, DRM voucher DNA1752 B Black form, Chile: Reg. X, Chiloé:
Rio Puntra at route 5, DRM voucher DNA2236 C Orange-spotted form, Argentina: Neuquén: Rio Pichi
Traful nr Lago Traful, DRM voucher DNA2564 D Orange-spotted form, Argentina: Neuquén: Rio
Pichi Traful nr Lago Traful, DRM voucher DNA2555. Scale bar 0.1 mm.

aspect of morphological or molecular variation was observed to be correlated with
presence or absence of the orange spot.

DNA sequence variation. As noted above under Results, there was minor varia-
tion present in COI and the wingless gene, and no variation in the other genes studied.

Habitat and seasonality. At all four localities where habitat data were recorded, B.
tetrapholeon specimens were found on cobble, gravel, and coarse sand shores of clear,
fast-flowing rivers (Fig. 6D), from 55 m elevation (Rio Puntra, Isla Grande de Chiloé,
Chile) to 830 m elevation (Arroyo Queni, Neuquén, Argentina). These shorelines lack
vascular plants. The beetles occur close to the water, most within 1 m. Specimens have
been found in January and February.

Geographic distribution. In southern Argentina and Chile (Fig. 18). In Argentina
this species has been found in Neuquén and Chubut, and in Chile from Regions X and XI.

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 147

Buenos Aires

specimens I have examined and that have been sequenced; circles filled in gray are based upon specimens
identified by Luca Toledano. Five cities are included as landmarks.

Relationship to other Bembidion. B. tetrapholeon is a member of subgenus Notho-
nepha, as strongly supported by DNA sequences (Table 3) and the presence of shared,
derived mesepisternal pits. B. tetrapholeon appears to be the sister of remaining Bembid-
ion (Nothonepha) (Figs 7, 8). Four genes support this placement (CAD, weg, ArgK, and
18S; Table 3), although the support is weak or moderate in single-gene analyses.

Concluding remark

“when the same organ appears in several members of the same class, especially if
in members having very different habits of life, we may attribute its presence to
inheritance from a common ancestor.” (Darwin 1859)

148 David R. Maddison / ZooKeys 416: 113-155 (2014)

In groups such as beetles, in which a preponderance of lineages split without later
reticulation, the evolutionary tree at the core of life’s history yields hierarchical pat-
terns in the distributions of characteristics. Any particular lineage in the tree, if sepa-
rated long enough or with a high enough rate of evolution, will leave in the bodies of
its descendants marks of its existence. The echoes from that deep historical well can
reverberate down through later lineages in the form of signals scattered throughout the
genomes. The repeated patterns of these branch markers both in the DNA and on the
bodies of organisms are among the most compelling signs of the existence of the tree
of life, and provide to us evidence about its shape. On occasion the clades thereby re-
vealed are so unexpected that it is only with multiple independent markers, all showing
the same pattern, that we can confidently accept the existence of the clade. Nothonepha
is such a clade.

Many of us who study the diversity of life, and see the hierarchical patterns of
organismal traits, are steeped in evidence of the existence of a genetic tree of life, so
much so that we perhaps take it for granted (I often do). I think about the evidence
about the tree’s shape, but much less so evidence about its existence. In encountering
the first evidence of Nothonepha, my belief in the tree-like structure of beetle genetic
history was challenged, as the data made little sense in that light. As newly sequenced
genes added to the evidence, my acceptance of the clade increased. The struggle was
fully resolved when the mesothoracic pits came to light; this harmonizing of the mor-
phological data with the molecular not only instilled a firm belief in this clade, but also
a simple confirmation of the tree itself.

Acknowledgements

Iam most grateful to Sergio Roig-Jufent, who arranged the collecting expedition that
yielded most known specimens of Bembidion tetrapholeon, and most of the other No-
thonepha studied; he was also a very welcome companion on the trip.

I am very thankful to all those who helped with a collecting expedition to Ecuador
during which most sequenced members of subgenus Ecuadion were collected. Mau-
ricio Vega arranged many details of the trip, including relevant collecting and export
permits; Wayne Maddison, Marco Reyes, and Mauricio Vega accompanied the author
into the field and helped collect the specimens. I am also grateful to Reserva Yana-
cocha, Pichincha, Ecuador, for permission to collect on their lands. The Ecuadorian
Ministry of the Environment and the Museum of Zoology of the Pontificia Universi-
dad Catélica de Ecuador assisted with permits.

I would also like to thank Elizabeth Arias and Kipling Will for organizing and
providing excellent logistical support, including permit acquisition, for field work in
Chile. I am also grateful for their companionship during my first experience with
South American Bembidion.

Some key specimens were provided by others, and I am thankful for their contri-
butions; without a network of collectors, a work such as this would not be possible. In

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 149

particular, I would like to thank William D. Shepard (for the Rio Pullinque specimen
of B. tetrapholeon), Wendy Moore (B. tucumanum), Karl M. Kjer (for South African
Tachylopha), Geoff B. Monteith (for Australian Tachylopha), Caroline S. Chaboo (for
South African Sphaerotachys), and James M. Pflug (for Oodinus from Texas).

Iam also grateful to Luca Toledano, both for his review of the manuscript, and for the
information he provided about specimens of B. tetrapholeon he has examined from Chile.
My thanks to Wayne Maddison and an anonymous reviewer for their comments on the
manuscript. Thanks as well to myrmecologists Alex Wild and Philip Ward for discussing
the ants that might live in the same habitats as Nothonepha, Tachylopha, and Oodinus.

This project was made possible by funds provided by the University of Arizona,
the Harold E. and Leona M. Rice Endowment Fund at Oregon State University, and
National Sciences Foundation grant EF-0531754. National Sciences Foundation DEB-
0445413, to Elizabeth Arias and Kipling Will, provided funds that helped support field
work in Chile. The expedition to South Africa that yielded the Elaphropus (Sphaerotachys)
I studied was supported by a Hellman Postdoctoral Fellowship (to C.S. Chaboo).

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Appendix |

David R. Maddison / ZooKeys 416: 113-155 (2014)

Locality data for newly sequenced specimens

Locality data for Bembidion specimens newly sequenced for this study. Under “#” the D.R. Maddison
DNA voucher number is listed.

# | Locality
B. agonoides 2675 |ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, 2090m, 0.6024°S, 77.8509°W
ares eae 2651 ECUADOR: Pichincha: Reserva Yanacocha, start Andean Snipe Trail, 3540m,
qe 0.1152°S, 78.5837°W
B. chimborazonum 2659 )|ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
pier mee 2658 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3460m,
Meee 0.1105°S, 78.5642°W
B. eburneonigrum 2204 |CHILE: Reg. IX: Rio Allipén at route 119, 132m, 39.0164°S, 72.5045°W
ARGENTINA: Neuquén: Rio Salado at route 40, 725m, 38.2143°S,
B. engelhardti 2334 70.0931°W
ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m,
B. georgeballi 2661 0.1105°S, 78.5642°W
B a 2336 ARGENTINA: Neuquén: Rio Salado at route 40, 725m, 38.2143°S,
. germainianum 70.0931°W
B. guamani 2660 |ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
B. humboldu 2673 |ECUADOR: Pichincha: Paso de la Virgen, 4070m, 0.3331°S, 78.2025°W
B. jimburae 2674 |ECUADOR: Napo: Rio Guango, 2730m, 0.3758°S, 78.0748°W, 26.x.2010
B. neodelamarei 2342 |ARGENTINA: Mendoza: Uspallata, 1880m, 32.5908°S, 69.3513°W, 25.ii-2007
B 2678 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m,
1 0.1105°S, 78.5642°W
B pauli i 2783 ECUADOR: Pichincha: Quebrada Lozada, on road to Res. Yanacocha, 3455m,
. paulinae paulinae 0.1105°S, 78.5642°W
B. philiopii 9327 ARGENTINA: Neuquén: Rio Collén Curd ca 13 km S La Rinconada, 625m,
gern 40.1015°S, 70.7545°W
B. ricei 2653 |ECUADOR: Napo: Rio Chalpi Grande, 2800m, 0.3645°S, 78.0852°W
B. sanctaemarthae 2652 |ECUADOR: Napo: Rio Chalpi Grande, 2780m, 0.3657°S, 78.0848°W
ECUADOR: Napo: Rio Cosanga at mouth of Rio Angenaro, 2185m, 0.6407°S,
B. sp. nr. caoduroi 2677 77 .9083°W
B. stricticolle 2240 |CHILE: Reg. IX: ca. 28 km E Melipeuco, 1262m, 38.83°S, 71.4038°W
CHILE: Region X, Rio Pullinque at Puente Huanehue, 8 km NE Panguipulli.
Ge ae 1752 | 39036°58"S, 72°13°43” W, 1590 fi.
B. tetrapholeon 2236 |CHILE: Reg. X, Chiloé: Rio Puntra at rt 5, 55m, 42.1661°S, 73.7256°W
ARGENTINA: Neuquén: Arroyo Quefi at Lago Quefi, 830m, 40.1575°S,
B. tetrapholeon 2356 71.7210°W
ARGENTINA: Neuquén: Arroyo Quefi at Lago Quefi, 830m, 40.1575°S,
B. tetrapholeon 2a57 71.7210°W
ARGENTINA: Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S,
B. tetrapholeon 2555 71.5958°W
ARGENTINA: Neuquén: Arroyo Quefi at Lago Quefi, 830m, 40.1575°S,
B. tetrapholeon 2562 71.7210°W
B. tetrapholeon 2563 |ARGENTINA: Chubut: Rio Azul at Lago Puelo, 200m, 42.0929°S, 71.6244°W
Bt tavavholson 2564 ARGENTINA: Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S,

71.5958°W

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 153

# | Locality

i wenolioleon 2565 Sela Neuquén: Rip Pichi Traful nr Lago Traful, 810m, 40.4867°S,
ee 2566 iSeaeea Neuquén: Arroyo Quefi at Lago Quefi, 830m, 40.1575°S,
Bt , 1430 ARGENTINA: Santa Cruz District, Dept. of Deseado, Cafadén Minerales. 25

Sites km S. of Caleta Olivia. 46.7146°S, 67.367°W. 25m.
B. walterrossii 2650 )ECUADOR: Napo: Vinillos, 4.1 km S Cosanga, 2090m, 0.6024°S, 77.8509°W
B. (Ecuadion) sp. 2701 ARGENTINA: Mendoza: Reserva Villavicencio, 1540m, 32.5232°S,
“Mendoza” 68.9949°W
Bt ougainny sp. 2657 |ECUADOR: Napo: Papallacta, 3315m, 0.3703°S, 78.1481°W

Papallacta
B. (Notholopha) sp. 2239

CHILE: Reg. IX: PN. Nahuelbuta, 1090m, 37.8274°S, 73.0096°W

“Nahuelbuta”

154 David R. Maddison / ZooKeys 416: 113-155 (2014)

Supplementary material |

NEXUS file containing DNA sequence matrices and maximum likelihood trees

Authors: David R. Maddison

Data type: NEXUS file with DNA data and trees

Explanation note: This is a NEXUS file containing the seven individual gene matrices
as well as the two matrices of the concatenated genes. In addition, it contains the
maximum likelihood trees for each matrix, and all of the bootstrap trees (2000 trees
for each matrix). The file is formatted to be opened in Mesquite 2.75 or later.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app1

Supplementary material 2

NEXUS file containing DNA sequences for B. tetrapholeon

Authors: David R. Maddison

Data type: NEXUS file with DNA data

Explanation note: This is a NEXUS file containing six matrices showing observed
sequences for all of the specimens of Bembidion tetrapholeon sequenced. ‘The file is
formatted to be opened in Mesquite 2.75 or later.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app2

An unexpected clade of South American ground beetles (Coleoptera, Carabidae, Bembidion) 155

Supplementary material 3

Images of maximum likelihood trees

Authors: David R. Maddison

Data type: images of phylogenetic trees

Explanation note: This file shows the maximum likelihood trees for the concatenated,
seven-gene matrices as well as each individual gene. Each figure is labeled to indi-
cate the nature of the data analyzed for that tree.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app3

Supplementary material 4

Images of maximum likelihood bootstrap trees

Authors: David R. Maddison

Data type: images of phylogenetic trees

Explanation note: This file shows the maximum likelihood bootstrap trees for the con-
catenated, 7-gene matrices as well as each individual gene. Each figure is labeled to
indicate the nature of the data analyzed for that tree. Numbers on each branch are
the frequencies of that clade in the bootstrap replicates, expressed as a percentage.

Copyright notice: This dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License
(ODDbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.

Link: doi: 10.3897/zookeys.416.7706.app4